Although hepatitis C virus (HCV) is a leading cause of morbidity and mortality worldwide, the effects of viral gene expression on infected cells remain unclear in vivo. Previously, we reported the construction of transgenic mice expressing HCV structural proteins (core, E1 and E2) and showed that expression of HCV structural proteins is not directly cytopathic in this animal model. Using DNA immunization, we were able to induce antibodies, T cell proliferative and cytotoxic T cell responses against the HCV core but only antibodies against the envelope protein in these transgenic animals. In contrast, the cellular immune responses appeared to target predominantly against the envelope proteins in wild-type mice. These results suggest a hierarchy of CTL response against the HCV structural proteins (envelope>core) in the wild-type mice, and an immunological ignorance toward the core but tolerance toward the envelope proteins at the T cell level in the transgenic mice. Our laboratory has also generated transgenic mice expressing HCV full-length polyprotein and is developing a system for the inducible expression of HCV transgene Based on the tetracycline regulatory system, we established a binary transgenic model in which the conditional expression of two transgenes, SV40 T antigen (TAg) and LacZ, can be tightly regulated in the liver by administration of tetracycline. Mice with tTA and TAg transgenes developed hepatocellular adenomas and hyperplasia that could be prevented by continuous tetracycline administration. This experiment demonstrates the value of this binary transgenic model in studying the physiological functions of any potential genes of interest in a liver-specific manner. We are currently evaluating the regulated expression of HCV proteins in the liver using this model system. We have also initiated studies in the chimpanzee which is the only infectious animal model for hepatitis C. We have constructed a molecular infectious clone of HCV genotype 1b and studied the detailed virologic, serologic, and immunologic parameters of four chimpanzees inoculated with this monotypic inoculum. All four chimpanzees developed minimal hepatitis on liver biopsy but no aminotransferase elevations were observed. Two chimpanzees developed persistent viremia, with titers of 103 to 105 genomes/ml. Sequence analyses of circulating viruses from both chimpanzees at various time points after infection demonstrated that relatively large proportion of mutations affecting protein sequences appeared in the NS5A gene, and 5 mutations were common to both chimpanzees. The appearance of a distinct pattern of mutations is suggestive of an adaptive response of HCV in vivo. Two of the chimpanzees cleared the infection to undetectable levels for 6 months of follow-up. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of IFN-gamma producing CD4+ and CD8+ T cells in peripheral blood by ELISpot did not reveal any correlation between viral clearance and T cell responses. In addition, analyses of IFN-gamma, IFN-alpha, and IL-4 mRNA levels in liver biopsies, presumably indicative of intraheptic T cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. Thus the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T cell responses. These animal models are valuable not only to address issues of immunopathogenesis and cytopathic potential of HCV gene products but also to study HCV replication in vivo.